LMP 301 Lecture 20: Molecular Diagnostics Flashcards
molecular diagnosis includes…
- pharmacogenetics
- identity testing (forensics)
- molecular genetics
- hematology
- molecular oncology
- infectious diseases
what is pharmacogenetics?
- personalized medicine
- prescribe drugs to person based on their genome
what type of disorder is sickle cell anemia (genetics)?
Autosomal recessive
what part of the genome has a problem in those with sickle cell anemia?
single point mutation in B-globulin gene
- E -> V
- restriction enzyme (MstII) is no longer able to recognize the sequence that it’s supposed to digest
MstII cuts between…
C and T
RFLP
restriction fragment length polymorphism
RFLP is used to…
- digest the genome with restriction enzymes
- use southern blot to detect length of digested pieces
- compare to normal to see if there is mutation / what the mutation affects
what differs in a mutant phenotype when looking at RFLP results?
usually, some sites are not cut (no longer recognized by the restriction enzyme). This will lead to larger segments when compared with the normal phenotype
How long does southern blot take?
about 1 week
process of southern blot (RFLP method)
- restriction enzyme digest sample
- electrophoresis on sample
- immobilization
- hybridization
- detection
limitations of southern blot based on RFLP method
- labour intensive
- time consuming / slow turn around time
- require operator skills / not automated
- use radioactive isotopes
steps of PCR
- denature DNA (95*C)
- anneal primers (55*C)
- extension - Taq adds nucleotides (72*C)
30 cycles of PCR can produce…
1 billion PCR products (new strands)
PCR allows us to look at target gene without…
highly sensitive probes (because there’s so many!)
turn-around time of PCR-RFLP
1-2 days
process of PCR-RFLP
- PCR reaction
- Restriction enzyme digestion
- Gel electrophoresis
limitations with PCR-RFLP
- based on known sequence (get right primer & restriction enzymes)
- risk of contamination (will amplify mistakes)
- miss heterozygous large insertion/deletion
what technique is used to examine multiple mutations associated with a disease?
multiplexed PCR-RFLP
multiplexed PCR-RFLP
- use multiple primers to amplify several DNA fragments in 1 run
- genotype based on electrophoresis pattern
what disease is genotyped using multiplexed PCR-RFLP?
Hereditary hemocromatosis (HH)
which gene is affected in those with HH?
HFE
mutations of CF
many mutations in the disease-causing gene
effect of CF / why is it named this way?
scarring (fibrosis) and cyst formation in the pancreas
what is the most common life-limiting autosomal recessive disease among Caucasians?
CF
what type of disease is CF (genetics)
autosomal recessive
incidence of disease
1 in 2500
incidence of carrier for CF
1 in 25
what is the affected gene in CF?
cystic fibrosis transmembrane conductance regulator (CFTR)
CFTR codes…
protein which is responsible for transport of Cl- ions across membrane (lungs, pancreas, liver, digestive tract, reproductive tract, skin)
CFTR is a ___ transporter
ABC (ATP-binding cassette)
what happens if there is defective CFTR?
thick, viscous, mucus secretions
onset of CF symptoms happen…
at birth or later in childhood
symptoms of CF
- frequent lung infection (due to mucus secretions)
- poor growth (malnutrition due to mucus in GI)
- infertility
- diabetes
CFTR has __ exons and span more than __ kb
27
230
CFTR gene is located on chromosome __
7
how many mutations are there for the CFTR gene?
more than 1600
purpose of genetic screening
diagnose affected patients and identify carriers
how many mutations CFTR mutations are screened for? why were they picked?
25; >0.1% frequency in US population
what are the most common mutations in CFTR?
F508: deletion for Phe at position 508
Platforms for CFTR mutations
- Tag-It mutation detection system
- Denaturing high performance liquid chromatography (DHPLC)
- non-PCR based technologies (Third Wave INVADER)
Tag-It mutation detection system
- bead-based microarray platform
- many primers that recognize specific sequences (mutations)
- catch about 50 mutations
- count beats to see if wild type or mutatnt
DHPLC
- adjust temp
- mismatches separate at lower temp than the right match
DNA sequencing
determines precise order of nucleotides in a DNA molecule
- any method/technology that determines the order of the bases
4 basic sequencing methods
- chemistry reaction
- technology
- labeling strategy
- sequencing detection
2 chemistry reactions used to sequence
- enzymatic dideoxy (Sanger)
2. chemical (Maxam-Gilbert)
2 technologies used to sequence
- chain termination
2. thermal cycle
2 labeling strategies used to sequence
- primers
2. dideoxynucleotides
2 sequence detection methods used to sequence
- radioactive
2. fluorescent
what is the gold standard for mutation detection & confirmation (sequencing)?
Sanger sequencing
Sanger sequencing
- use DNA Pol to make a copy of ssDNA template at the 3’ end of a primer
- use PCR
- terminate by adding 2’, 3’-dideoxynucleotides
- analyze fragment using gel/capillary electrophoresis
benefits of Sanger DNA sequencing
- automated (capillary electrophoresis)
- CCD laser detector
- multicolour fluorescent labeling & detection
- many applications for data (sequencing, fragment analysis, genotyping & SNP discovery, microsatellite analysis)
- software for data collection & analysis
how does Sanger sequencing method terminate?
Add 2,3-dideoxyribose dideoxynucleodies
- li H instead of OH at C3
- won’t H bond with the next nucleotide, so chain stops
limitations of Sanger sequencing
- miss heterozygous large insertion/deletion
- cost, labour, turn-around time
- difficult to interpret variants with unknown clinical significance (variation in genome or harmful mutation?)
clinical applications of DNA sequencing
- detect mutations
- confirm mutations detected by other methods
- genotyping (histocompatibility typing, resistance testing)
histocompatibility typing
used for transplant patients to see if organ is compatible
